Nuclear magnetic resonance (NMR) spectroscopy is a versatile analytical technique used in a broad range of disciplines, ranging from materials science, chemical synthesis, catalysis, structural and cell biology, oil-well logging, metabolomics and metabonomics, and in vivo spectroscopy and imaging. Its great strength resides on its site selectivity and sensitivity to changes in local environments. Experiments can be tailored to monitor specific aspects of the molecules under study, such as the local electronic environment, the topology, distances and angles in molecules, their motional properties, and intermolecular interactions.
The study of naturally occurring biomolecules with NMR has always been of great interest to elucidate structure, reactivity and properties of functional groups in peptides. However, with large molecules, interpretation of NMR spectra may be difficult, in particular for the analysis of specific fragments. In peptides or proteins, the capability of visualizing sites of interest is reduced due to overlapping resonances or reduced intensity. Multidimensional NMR experiments are often used to discriminate these sites from the rest of the molecule, but sometimes the spectrum can still contain obscured peaks and a complete characterization is difficult. Isotopic labeling methods are also utilized; however, the labeling process may be complicated for naturally occurring biomolecules. An alternative method to make the peaks more prevalent is by using a hyperpolarized label.
Many hyperpolarization techniques are being used to study biomolecules such as hyperpolarized Xe for the study of protein interactions and binding dynamics, and exploiting the difference in chemical shift of the gases. Dynamic nuclear polarization (DNP) methods including (photo-) chemically induced and high-field experiments are used to obtain information including structural features, and folding dynamics of proteins. Although these methods are highly effective, the experiments involve complicated setups and difficult implementation.
The phenomenal site-selectivity exhibited by NMR spectroscopy is also related to one of its principal drawbacks—detection sensitivity. Several avenues for improving sensitivity have been identified, including dynamic nuclear polarization, optical pumping, and parahydrogen (p-H2) induced polarization (PHIP) techniques. Over the last two decades, prior art PHIP experiments including their specific implementations in the form of the well-known PASADENA and ALTADENA work, have been developed to improve the sensitivity of NMR by a factor of 104 to 105. The polarization can be incorporated into the molecular structure in two ways: 1. The SABRE method where the polarization is exchanged from the para-hydrogen to the target molecule via a catalytic intermediate; or 2. Through a hydrogenation reaction. Although the former method achieves polarization transfer without changing the chemical structure, it may not be easy to select specific sites and has only been shown to be effective on relatively small specific molecules. Hydrogenation with p-H2, i.e. ALTADENA and PASADENA experiments, are fairly easy to implement and can be performed on any sample possessing an asymmetric multiple bond. One of the limitations of any form of proton polarization enhancement is that the signal will last only as long as typical T1 relaxation times in the molecules. This makes them difficult to implement for some experiments such as diffusion experiments and magnetic resonance imaging using hyperpolarized contrast agents, because they may require many seconds for molecular distribution within the system to occur. Bargon et al. have shown that the lifetime of the hyperpolarization in low magnetic fields can exist for 300 s.
Carravetta et al. and others in the prior art have shown that one can store the nuclear spin order using low field nuclear spin singlet states for times much longer than the T1 relaxation time constants. However, such ingenious approaches will be practical only for a select subset of systems and they require quick field cycling between high and low magnetic fields. The long longitudinal relaxation time of 13C nuclei has also been exploited for long lived polarized signals. This nucleus provides a means for storing the polarization for longer times, since T1 of 13C is typically much longer than for 1H spins. However, with the extremely low natural abundance of 13C (<1.1%), enriched compounds are required, and the efficient transfer will very much depend on the coupling patterns within the molecules.
Another limitation is that the high polarization can typically only be used in conjunction with small flip angle pulses, otherwise the available signal quickly dephases on the order of T2. With a large flip angle, the polarization would have to be replenished with p-H2 between one experiment and the next.
Therefore, the benefit of further extending the time within which hyperpolarized 1H signals could be observed would provide a broad range of new applications.